Substituted and unsubstituted vinyloxycarbonyl groups as amino protecting groups in the syntheses of peptides

ABSTRACT

Temporary blocking, in peptide syntheses, of a free amino group by acylating same and, after peptide condensation, splitting off the acyl group introduced, wherein the free amino group is acylated with a substituted or unsubstituted vinyloxycarbonyl group is disclosed. The vinyloxycarbonyl group may be removed from a product peptide with bromine or another halogen followed by alcohol, with a mild acid or by mercuric ion induced hydrolysis.

United States Patent [191 Olofson et al.

[ 1 Jan. 16, 1973 [54] SUBSTITUTED AND UNSUBSTITUTED VINYLOXYCARBONYLGROUPS AS AMINO PROTECTING GROUPS IN THE SYNTHESES OF PEPTIDES [75]Inventors: Roy A. Oloison; Yasushi Stephen Yamamoto, both of StateCollege,

[73] Assignee: Research Corporation, New York,

[22] Filed: Aug. 21, 1969 21 Appl. No.: 852,096

[52] US. Cl. ..260/l12.5, 260/309, 260/326.14,

260/326.3, 260/471, 260/481, 260/482 C [51] Int. Cl. .....C07c 103/52,C07g 7/00, C08h l/OO,

5/1969 Sakakibara et al .260}! 12.5 2,723,972 ll/l955 Herrick et al...260/l 12.5

OTHER PUBLICATIONS Southard et al., Tetrahedron Letters 3505-3508.Grimshaw, J. Chem. Soc. 1965, 7136-7139.

Stevens et al., J. Am. Chem. Soc. 72, 725-727 (1950).

Boissonnas et al., Helv. Chim. Acta 36, 875-886 (1953).

Primary ExaminerElbert L. Roberts Attorney-Holman and Stern [5 7]ABSTRACT Temporary blocking, in peptide syntheses, of a free amino groupby acylating same and, after peptide condensation, splitting off theacyl group introduced, wherein the free amino group is acylated with asubstituted or unsubstituted vinyloxycarbonyl group is disclosed. Thevinyloxycarbonyl group may be removed from a product peptide withbromine or another halogen followed by alcohol, with a mild acid or bymercuric ion induced hydrolysis.

6 Claims, No Drawings SUBSTITUTED AND UNSUBSTITUTED VINYLOXYCARBONYLGROUPS AS AMINO PROTECTING GROUPS IN THE SYNTHESES OF PEPTIDES Theinvention described herein was developed under a grant (GM-13980) fromthe U.S. Public Health Service, Department of Health, Education andWelfare.

This invention relates to novel stable vinyloxycarbonyl derivatives ofamino acids, peptides and amino acid and peptide derivatives all usefulas reagents or intermediates in peptide syntheses and also to processesfor using the same in the syntheses of peptides. In particular thisinvention provides methods for the temporary blocking in peptidesyntheses of a free amino group by acylating it with a new protectinggroup the vinyloxycarbonyl group and after peptide condensation has beenaccomplished, splitting off the vinyloxycarbonyl group introduced.

One of the major problems in peptide syntheses is the efficientprotection of an a-amino function in an amino acid or peptide whilecoupling the protected product at the carboxyl end thereof with an aminoacid or peptide or derivative thereof exhibiting a free a-amino group toform an amide bond. In peptide syntheses the protection or blocking ofthe amino group of one of the amino acids or peptides involved in thereaction followed by t-butoxycarbonyl (hereinafter tB O C) II 1T1 02 So-nitrophenylsulfenyl (hereinafter N PS") III 5 The above prior artblocking groups are all ordinarily introduced by treatment of the aminoacid with an active acyl form of I, II or III (Cbz chloride, tBOC azide,NPS chloride) in base. They are all usually best removed from a productpeptide by treatment with an acid, either I-lBr or often, with II orIII, the milder HCl.

However when the performance of each of the prior art blocking groupsreferred to above is measured against the complexity of presentsynthetic objectives in peptide chemistry it is obvious there is greatneed for a better blocking group one which may be removed in higheryield and under milder conditions than these species and one which maybe removed under conditions so selective that the C82, tBOC and NPSresidues are unaffected. Fulfillment of this final requirement wouldallow the use of these latter blocking groups for the protection ofsecondary functions in a synthetic scheme. The groups now used for thispurpose benzyl, tosyl, etc. are so stable they can sometimes be detachedonly under conditions in which the product peptide is itselfdismembered.

, Thus, it is a primary object of the instant invention to provide a newblocking or protecting group and 1 earbobenzoxy (hereinafter Cbz) Iprocedures for using same in peptide syntheses, which are free from theforegoing and other such disadvantages. In this regard, it is animportant object of the instant invention to provide a blocking groupwhich may be removed in higher yield and under milder conditions thanthose blocking groups presently known to the art. Moreover, it is abasic object of the instant invention to provide a blocking group whichmay be removed under conditions which are so selective that the priorart blocking groups, Cbz, tBOC, and NPS, are unaffected therebypermitting the use of these latter groups for the protection ofsecondary functions.

It is another important object of this invention to provide a blockinggroup for the a-amino function in peptide chemistry which is extremelyinexpensive, particularly when compared with prior art materials,whereby use of the new blocking group is quite economical. Additionally,the instant invention contemplates the provision of a new blocking groupfor peptide syntheses which may be readily added to the free amino groupof an amino acid or peptide material and which may be readily removedfrom a product after peptide condensation.

The foregoing and other such desiderata may be realized by the use ofthe vinyloxycarbonyl group:

i CH2=CH O C- vinyloxycarbonyl group (hereinafter VOC) Vinylchloroformate is a known compound readily synthesized in 40-45 percentyield by passing the bischloroformate of ethylene glycol through a hottube.

Details of the production of vinyl chloroformate can be found in U.S.Pat. No. 2,377,085 dated May 29, 1945 and in a paper by L.-H. Lee (J.Org. Chem., 30, 3943 (l965)).

Although vinyl chloroformate is the preferred material, other sources ofthe VOC group may be utilized according to the instant inventiveconcepts. For example, other vinyl haloformates and in general species,CH =CHOCOX, where X is a good leaving group may be utilized in place ofvinyl chloroformate.

In addition substituted vinyloxycarbonyl groups should also be useful astemporary protecting groups in peptide syntheses according to instantinventive concepts. These can be introduced by treatment of an aminoacid or peptide with a substituted vinyl haloformate or X-formate andremoved in the same way as the vinyloxycarbonyl group has been removed(see below). Thus, the term vinyloxycarbonyl group as utilizedthroughout this specification and in the appended claims shall beunderstood to include substituted vinyloxycarbonyl groups and may berepresented by the following general formula:

wherein R, R and R may be the same or different and may be H, alkyl,aryl, alkoxy, acyloxy, carboalkoxy, carboxamido, acyl, or secondaryamino groups. R and R and R may be joined in a ring (except that R and Rmay not be joined as a benzene or substituted benzene ring). The usualfurther substituents used as an aid in obtaining greater crystallinity(i.e. S NR may serve as part of R, R or R Several techniques arearailable for removal of the VOC group from a product after peptidecondensation. For example, the VOC group may be split off by titrationof the resultant VOC peptide with bromine in an inert solvent followedby the addition of methanol and the removal of the solvent and the BrCHCl-flOMe) at reduced pressure to produce the peptide hydrobromide.Alternatively the VOC group may be split off by treatment of theresultant VOC peptide with a mild acid. Moreover, the VOC group may besplit off from the VOC peptide by mercuric ion induced hydrolysis.

In order to facilitate an understanding of the instant inventiveconcepts, a definition of some of the terms used herein will be helpful.

The term temporary blocking or temporary protection shall be understoodto mean the same as in US. Pat. No. 3,304,296 (R'. Schwyzer, K. Zatsko,P. Sieber, and H. Kappeler): that, in the synthesis of peptides, in astarting amino acid or peptide or a carboxylic acid derivative thereof,a free amino group which must not participate in the condensationreaction is protected, for instance by acylation, the thus-protectedproduce coupled at the carboxyl end with an amino acid or peptide orderivative thereof exhibiting a free 0:- amino group to form an amidebond, and that from the I condensation product the protecting group isremoved.

It is possible, of course, to use the condensation product still havingthe amino group protected as starting material for further condensationreactions with amino acids or peptides as are customary in the synthesisof higher peptides (being built up from a large number of amino acids)and to remove the protective group only at the end of the synthesis.

The carboxylic acid derivatives mentioned above are those which areknown to be useful in peptide syntheses for carrying out thecondensation reaction. Thus, for instance, the amino acid or peptidewhich is to be coupled at the carboxyl end'with another amino acid orpeptide is used in the form of the free acid when the carbodiimidemethod isused for condensation, or in the form of the azide, when theazide method is used, or in the form of a carboxylic acid or carbonicacid mixed anhydride when the mixed anhydride method is used, or in theform of a reactive ester, viz. a methyl or phenyl ester substituted byan electron-attracting substituent that is to say a meta-directingsubstituent, for instance cyanomethyl ester, ca'rbethoxym'ethyl ester,or para-nitrophenyl ester. On the other hand, the amino acid or peptidewhich is to be coupled by means of the free group can be any carboxylicacid derivative, for instance a lower alk'yl or phenyl lower alkyl,especially a benzyl, para-nitrobenzyl, para-halogenobenzyl or paraloweralkoxybenzyl ester, or an activated ester as defined above or an amide,hydrazide or anhydride. It should be noted that the kind of carboxylicacid derivative and the manner of condensation are immaterial withregard to the protection of the amino group involved here. Also, it isimmaterial whether the amino acids or peptides to be condensed containfurther protected functional groups, for instance an acylated hydroxygroup.

By the term amino acid in this application shall be understood to meanthe so-called natural amino acids, viz. those amino acids which areformed in the hydrolysis of naturally occurring peptides and proteins,for instance glycine, alanine, valine, leucine, norleucine, cysteine,methionine, omithine, lysine, arginine, aspartic acid,asparagine,.glutamic acid, glutamine, proline, hydroxy-proline, serine,threonine, histidine, tryptophane, phenylalanine, tyrosine and othera-amino acids. The amino group to be protected is the a-amino group oran amino group present in the side chain for instance the e-amino groupof lysine. The amino group to be protected is a primary amino groupexcept in the case of proline or hydroxyproline where the a-amine groupisa secondary amino group. If desired the aamino group and any aminogroup of the side chain may be protected by the same protecting group.As to the configuration of the amino acids defined it can be the L- orD-configuration, the configuration being irrelevant in the protection ofthe amino group.

The term amino acid derivative shall be understood to mean substancesderived from amino acids which are ordinarily used as intermediates inthe synthesis of peptides. These include compounds where the carboxylgroups are present as salts (such as with metal cations or amines),where carboxyl groups are masked as esters or active esters (i.e.O-methyl, O- ethyl, O-t-butyl, O-benzyl, O-pNO -phenyl etc.), amides,hydr'azides, azides, anhydrides, or mixed anhydrides; and where otheramine, alcohol, sulfhydryl, or phenolic groups are protected with groupssuch as carbobenzoxy, carbo-t-butoxy arylsulfenyl, benzyl, tosyl,trityl, trifluoroacetyl, etc.

The term peptide shall be understood to mean peptide built up from aminoacids as defined.

The term peptide derivative shall be understood to 'mean peptidecontaining the substitutions described under amino acid derivative.

The products obtained .when amino acids, peptides or protectedderivatives thereof are acylated with a source of the VOC groupaccording to this invention are valuable reagents and intermediates inthe synthesis of various peptides, compounds containing peptidicfragments and proteins of known utility such as, for example in themanufacture of synthetic peptide hor mones. Natural peptidic materialswith, important biological and pharmaceutical activity include ACTH,aand B-MSH, g'rarnicidin, tyrocidin, actinomycin, bradykinin, oxytocin,vasopressin, and insulin. Further synthetic analogues of some of theabove including especially ACTH have been shown to have valuablepharmaceutical activity. Synthetic peptides related to ACTH are now soldcommercially in Europe (and probably also soon in the US.) and aretaking over an expanding share .of the world anti-inflammatory market.Further it is generally anticipated by workers in this field thatadditional (though possibly yet unknown) synthetic peptides will becomevaluable pharmaceuticals and much effort is now being expended aroundthe world in this area. Any procedure which allows the more economicalsynthesis of known (or soon to be known) peptide pharmaceuticals thushas tremendous commercial potential. It is believed that the instantinvention describes such a procedure.

Production of the VOC amino acids or peptides is readily accomplished inyields averaging 40-95 percent by treatment of a naturally occurringamino acid or peptide with a source of the-VOC residue, such as vinylchloroformate in base using procedures related to those previouslyworked out for the introduction of other prior art acyl protectinggroups. In a typical procedure the amino acid or peptide or protectedderivative thereof is dispersed in water and cooled in an ice bath.Vinyl chloroformate in dioxane is slowly dripped into the reactionvessel with stirring and sufficient magnesium oxide is also added duringthis period to keep the solution basic. After the addition is completedthe mixture is stirred for 4 hours at room temperature and then workedup by acidification with citric acid followed by extraction with ethylacetate. In those cases where the acid itself has not yet crystallizedthe product may be isolated and characterized as the dicyclohexylamine(hereinafter DCHA) salt as is commonly done with some of the tBOC-aminoacids. N-e-VOC-L-lysine is synthesized by reaction of the copper complexof lysine with vinyl chloroformate in aqueous base.

The VOC group is stable under standard peptide coupling conditionsusing, for example, dicyclohexylcarbodiimide (hereinafter DCC) orN-ethyl-S-phenylisoxazolium-3-sulfonate (hereinafter NEPlS) as the amidebond forming reagent. VOC-L-phenylalanyl- L-leucine methyl ester isobtained, for example, in 80 percent yield using DCC as the couplingreagent, while VOC-L-asparaginyl-glycylglycine ethyl ester is isolatedin 85 percent yield using the Woodward-Olofson reagent, NEPIS, as thecoupling agent. In general, peptide yields from VOC amino acids are thesame as those obtained for similar non-VOC peptides using the samepeptide forming reagent.

VOC-L-prolylglycine acid is prepared by base hydrolysis of the esterwhereby it appears that the VOC group is stable enough to base to allowhydrolytic operations at the carboxyl end of the peptide, a very usefulproperty. VOC peptides themselves are stable for long periods of time.These compounds may, however, be induced to undergo photochemical orradical polymerization yielding some potentially interesting substances.

Removal of the protecting group after peptide condensation may bereadily accomplished by several methods as mentionedpreviously.

For instance when VOC-diglycine ethyl ester is titrated with bromine inmethylene chloride, the dibromide from this highly activated double bondinstantaneously precipitates out. When methanol is added to the reactionmixture the dibromide immediately dissolves and carbon dioxide isevolved. After a few minutes the reaction mixture is evaporated todryness leaving diglycine ethyl ester hydrobromide. In this case thereaction is so clean that the residue from the evaporation has the samemelting point as the pure dipeptide ester hydrobromide prepared byclassical methods. The general reactions are diagrammed below:

BrCHzCH(0Me)2 C0; HBrJhN-peptldo Other halogenation reagents (Cl lpseudohalogens) will also work in this reaction and other alcohols maybe used.

In order to show that the tBOC group is stable under these conditions,N-e-tBOC-N-a-VOC-lysine was treated with bromine and methanol andN-e-tBOC- lysine was rapidly and cleanly obtained. Ordinarily, thesereactions are carried out without direct isolation of the dibromide andcan even be run with bromine in alcohol directly. Using one equivalentof Br there is no complication with halogenation of the aromatic ring ofphenylalanine.

As indicated, the VOC group may also be removed by the use of a mildacid (for example: HCl, HBr, or trifluoroacetic acid in alcohol, dioxaneor other inert solvent), again in a reaction which is quite clean andqualitatively occurs under the same conditions and at about the samerate as removal of the tBOC group. The tBOC residue is the most widelyused protecting group in peptide chemistry today. Since the VOC groupcan be removed with approximately the same yields and under the sameconditions as tBOC, and since vinyl chloroformate can be made at about 5percent of the cost of t-butoxycarbonyl azide it would appear that theVOC group has great potential in peptide syntheses with the acid inducedhydrolysis being the most widely used method for its removal.

Additionally, as mentioned, the VOC group may be removed by mercuric ioninduced hydrolysis, the use of mercuric acetate in acetic acid at 50 Cbeing found to deprotect VOC phenylalanine in about 10 minutes. Themercuric ion is easily removed by precipitation with hydrogen sulfide.

Finally it is anticipated that the vinyloxycarbonyl group will be splitoff by hydrogenolysis in the presence of appropriate heavy metalcatalysts though in particular cases and with certain catalystshydrogenation of the double bond may be an important side reaction.

The following examples are given as illustrative of the instantinventive concepts:

EXAMPLE I Vinyloxycarbonylglycine: Method A. Magnesium Oxide Procedure A50 ml three-necked round bottom flask, charged with glycine 1.5 g, 0.02M) in 15 ml water, is equipped with a magnetic stirring apparatus and adropping funnel containing vinyl chloroformate (3.2 g, 0.03 M) in 8 mldioxane. The flask is immersed in an ice bath and the solution stirredrapidly. Magnesium oxide powder (1.2 g, 0.03 M) is added in threeportions while the chloroformate is dripped in (30 minutes). The icebath is then removed and the solution stirred for an additional fourhours at room temperature. The reaction mixture is next cooled to C,acidified to Congo Red with 6N hydrochloric acid, and finally extractedwith three 50 ml portions of ethyl acetate. The combined extracts arewashed with two 25 ml portions of water, dried over anhydrous sodiumsulfate and the solvent stripped off under vacuum. The solid residue,vinyloxycarbonyl-glycine, is recrystallized from chloroform. Yield 2.35g, 81%. M.P. 94.5 95.5. Anal. Calcd. for C H-,NO C, 41.38%; H, 4.86%; N,9.65%. Found: C, 41.53%; H, 4.64%; N, 9.48%.

By a similar procedure the following vinyloxycarbonyl derivatives(abbreviated VOC derivatives) can be synthesized (Table I):

VOCL-alanine VOC-L-aspartic acid-fi-methyl ester VOC-S-benzyl'L-cysteineVOCL-glutamine VOC-L-glutamic acid-'y-methyl ester VOC-L-leucineN-a-VOC-N-e-tBOC-L-lysine VOC-L-methionine voGL-phenylalanineVOC-L-serine VOC-L-tyrosine VOC-L-valine obtained as oil* obtained asoil" obtained as oil obtained as oil* obtained as oil obtained as oilobtained as oil obtained as oil obtained as oil obtained as oil*obtained as oil* obtained as oil VOC-glycine ethyl ester liquid b.p. 72at 0.2 mm.

*See further characterization as DCHA salt in Table lll in Example 4.

EXAMPLE 2 Vinyloxycarbonylglycine: Method B. pH Stat Procedure Athree-necked, 100 ml round bottom flask is equipped with a droppingfunnel, mechanical stirrer, pH electrode and a neoprene delivery tube,running from near the electrode, through an automatic titration unit toa 50 ml burette containing ca 6N sodium hydroxide. (Each drop of basewhich is added by the titrator must fall right next to the electrode sothat the end point is not overshot due to the delay caused in mixing).The flask is charged with a solution of glycine (7.51 g, 0.1 M) in 20 mlof 50% dioxane/water and is immersed in an ice bath. The pH stat is setat pH 9.010.] and the solution is stirred vigorously'as vinylchloroformate (12.8 g, 0.12 M) in 10 ml dioxane is added dropwise overaminute period through the dropping funnel. The pH stat stops admittingbase to the system after another five minutes. After an additionalfifteen minutes in the ice bath, the reaction mix-- ture is acidified topH 4 with solid citric acid and then extracted with six 50 ml portionsof ethyl acetateyThe combined extracts are washed with water, dried overanhydrous sodium sulfate and evaporated to dryness under reducedpressure. The resulting solid is recrystallized from chloroform,yielding 9.1 g (75%) of vinyloxycarbonylglycine. M.P. 94.5-95.0. Secondcrop, 1.5 g, M.P. 94.0-95.0.

By a similar procedure the following vinyloxycarbonyl (VOC) derivativescan be prepared (Table II):

TA B LE ll Compound crystallized from M.P. VOC-L-asparagine ethylacetate- 15 7-! 5 9 pentane VOC-L-tryptophane ether-pentane 67 .5 -70VOC-L-alanine obtained as oil VOCL-hydroxyproline obtained as oilVOC-L-isoleucine obtained as oil VOC-L-leucine obtained as oilVOC-L-methionine obtained as oil VOC-L-phenylalanine obtained as oilvVOC-L-serine obtained as oil' VOC-L-threonine obtained as oilVOC-L-valine obtained as oil See further characterization as DCHA saltin Table Ill in Example 4.

EXAMPLE 3 Vinyloxycarbonylglycine: Method C. Triethylamine Procedure A250 ml three-necked round bottom flask, immersed in an ice bath, isequipped with two dropping funnels and a magnetic stirring bar. Intothis flask is placed a solution of-glycine (3.76 g, 0.05 M) in l00 ml of25 percent dioxane/water. The solution is rapidly stirred as vinylchloroformate (8.0 g, 0.075 M) in 25 ml dioxane is dripped in throughone dropping funnel while triethylamine (7.59 g, 0.075 M) issimultaneously added through the other. The addition rates are adjustedto keep the mixture basic at all times. After 30 minutes, the additionis completed. The ice bath is removed and the solution stirred for threehours at room temperature. It is then cooled again in an ice bath,acidified with 6N hydrochloric acid to pH 3 and the solvent evaporatedunder vacuum. The solid residue is extracted with 200 ml ethyl acetate,filtered and the filtrate washed with water and dried over anhydroussodium sulfate. The ethyl acetate is removed in vacuo, and the resultingvinyloxy-carbonylglycine is recrystallized from chloroform. Yield 1.5 gor 21 percent.

EXAMPLE 4 Vinyloxycarbonyl-L-phenylalanine Dicyclohexylamine Salt (It isoften useful to further characterize those vinyloxy-carbonyl amino acidsobtained as oils or amorphous solids as crystalline salts. Since theVOC- amino acids are easily and cleanly regenerated from salts bytreatment with acid followed by extraction into an organic solvent,these salts are sometimes useful in the further purification of VOCamino acids).

Dicyclohexylamine (4.6 g, 0.25 mol) is added dropwise to a cooledsolution of vinyloxycarbonyl-L-phenylalanine (4.7 g, 0.02 mol) in 50 mlether. During the addition (or sometimes later) a white solid slowlycrystallized out. This is filtered, washed with ether and thenrecrystallized, from absolute ethanol-ether. Yield: (97%); M.P. l58l59.Anal. Calcd. for C I-1 N 0 C, 69.18%; H, 8.72%; N. 6.73%.

Found: C, 69.37%; H, 8.36%; N, 7.06%.

TABLE III Dicyclohexylamine Salt from: crystallized from: M.P.VOC-L-alanine ethanol-ether l52.5 l 5 3 VOCL-aspartic acid-B-methylester ethanol-ether pentane 141 .5l43 VOC-S-benzyl-L-cysteinemethanol-ether pentane 143l44 VOC-L-glutamine ethanoletherpentanel47.5l49.5 VOC-L-glutamic acid-ymethyl ester ethanol-ether l55- 56VOC-L-hydroxyproline ethanol-ether I79.5-l ii I .5" VOC-L-isoleucinemethanol-ether l-l 8 l .5 VOC-L-leucine methanol-ether l57.5-158N-aVOC-N-e-tBOC-L-lysine ethanol-ether l53l54 VOC-L-methionineethanol-ether l 36138 VOC-L-serine ethanol-ether 218 (dec)VOC-L-threonine ethanol-ether 165 (dec) VOC-L-tyrosine methanol-ether190-195 (dec) VOC-L-valine ethanol-ether l 58-l 60 EXAMPLE 5N-e-Vinyloxycarbonyl-L-lysine A solution of 10.0 g (0.055 g) L-Lysinemonohydrochloride in 80 ml water is treated with g of a 2:1 CuCO /Cu(OH)mixture and boiled for 30 minutes. The hot solution is filtered toremove the precipitated cupric carbonate which is washed with 10 ml hotwater. The filtrate is cooled in an ice bath and 3 g magnesium oxideis'added. This mixture is then stirred rapidly while a solution of vinylchloroformate (8.52 g, 0.08 M) in 150 ml dioxane is added dropwise overa period of one hour. Stirring of the dark green mixture is continuedovernight at 45 C. The solution is next cooled in an ice bath, acidifiedwith 50 ml 2N acetic acid, and stirred for 1 hour. The precipitatedcopper complex is then filtered and washed with cold water and methanol.In order to free the N-e-VOC'L- lysine from the copper, the complex issuspended in 300 ml boiling water; acetic acid ml) is added, immediatelyfollowed by thio-acetamide (7.5 g, 0.1 M). The solution is boiled for anadditional ten minutes, while the cupric sulfide precipitates. Noritdecolorizing charcoal is added and the hot mixture is quickly-filteredon a Buchner funnel which has been covered by a mat of analytical filteraid. The filtrate is concentrated under vacuum to a volume of about 100ml, cooled in the refrigerator to crystallize the N-e-VOC-L-lysine andfiltered. The white solid is washed with a little cold water and driedin vacuo over potassium hydroxide. Yield 4.1 g, 34.5 percent (based onL-lysine monohydrochloride). M.P. 212 (dec).

EXAMPLE 6 Vinyloxycarbonyl-L-asparaginylglycyl-glycine EsterN-Ethyl-S-phenylisoxazolium-3'-sulfonate (2.026 g, 8mM) and 40 mlacetonitrile are placed in an ice cooled 125 ml Erlenmeyer flask andstirred with a magnetic stirring apparatus. A solution of vinyloxycarbomyl-L-asparagine (1.617 g, 8mM) and triethylamine (0.8l0g, 8mM) in 12acetonitrile is added and stirring continued until the reaction mixturehas become clear yellow (about 45 minutes). Glycylglycine ethyl esterhydrochloride (1.558g, 8mM) is then added, immediately followed by asolution of triethylamine (0.8lg, 8mM) in acetonitrile. The reactionmixture is stirred overnight at room temperature. Next the solvent isstripped of? at reduced pressure and the residue triturated with hotwater. The tripeptide crystallizes upon cooling, and is recrystallizedfrom ethanol/water. Yield 2.34g (85 percent), M.P. l86-l89. Analyticalsample M.P. l90.5-l92.0.

Examples of peptides which can be prepared by similar procedures arevinyloxycarbonylglycylglycine ethyl ester (M.P. 1l5-1l5.5),vinyloxycarbonyl-L- prolylglycylglycine ethyl ester (M.P. l45.5-l46.5),vinyloxycarbonyl-L-phenylalanylglycine ethyl ester (M.P. 124-l26), andvinyloxycarbonyl-L-asparaginyl-L-seryl-L-phenylalanyl-L-leucine methylester (M.P. =200 amorph).

Ethyl EXAMPLE 7 Vinyloxycarbonyl-L-leucyl-L-leucine Methyl Ester Amixture of vinyloxycarbonyl-L-leucine dicyclohexylammonium salt (3.83g,0.01 M), L-leucine methyl ester hydrochloride (1.82g, 0.01 M) and 60 mlchloroform are stirred while N,N-dicyclohexyl-carbodiimide (2.3g, 0.011M) is added. Stirring is continued for three hours. The precipitateddicyclohexylamine hydrochloride and dicyclohexylurea are filtered offand the filtrate washed with successive portions of water, dilutesulfuric acid, sodium bicarbonate and again with water; dried overanhydrous sodium sulfate, and finally evaporated to dryness in vacuo.Ethyl acetate is added to the residue and some undissolved urea filteredoff. The filtrate is evaporated to givevinyloxycarbonyl-L-luecyl-L-leucine methyl ester, which is thentriturated with hot methanol and crystallized in the refrigerator. Yield1.60g, M.P. 92-94-; second crop 0.43g, M.P. 9l.5-93 (overall 62%).Analytical sample, M.P. 9394. Anal. Calcd. for C16H26N205; C, 58.51%; H,8.59%; N, 8.53%. Found: C, 58.83%; H, 8.46%; N, 8.71%.

Examples of peptides which can be synthesized by similar methods arevinyloxycarbonyl-L-alanylglycine ethyl ester (M.P. l8l-183),vinyloxycarbonyl-L- methionylglycine ethyl ester (M.P. 105106),vinyloxycarbonyl-L-phenylalanyl-L-leucine methyl ester (M.P. l04-l05),and vinyloxycarbonyl-L-glutamyl-(F-methyl ester)-glycine ethyl ester(M.P. 86.5).

EXAMPLE8 Vinyloxycarbonyl-L-prolylglycine Free Acid and DCHA Salt A 25ml Erlenmeyer flask containing N-ethyl-S- phenylisoxazolium-3-sulfonate(1.0l2g, 4mM) in 10 ml acetonitrile is placed in an ice bath. Themixture is vigorously stirred with a magnetic stirring apparatus and asolution of vinyloxycarbonyl-L-proline (0.741 g,

4mM) and triethylamine (0.41g, 4mM) in 6 mlacetonitrile added. After 60minutes 0.560g (4mM) glycine ethyl ester hydrochloride and triethylamine(0.4lg, 4mM) in acetonitrile are also added. The mixture is stirredovernight at room temperature, then evaporated to dryness in vacuo andthe resulting residue partitioned between water and ethyl acetate. Thelayers are separated and the aqueous phase extracted twice with ethylacetate. The organic layers are combined, dried over sodium sulfate, andthe ethyl acetate stripped off at reduced pressure yielding an oil whichis hydrolyzed in methanol/dilute aqueous sodium hydroxide. The residueobtained on removal of the solvent at reduced pressure is dissolved indilute sodium bicarbonate solution. This is then acidified with solidcitric acid and extracted with ethyl acetate. The organic phase is driedover sodium sulfate and the solvent removed at reduced pressure. Theoily dipeptide acid is converted to its DCHA salt by the usualprocedure; recrystallized from acetone-pentane; M.P. l6l-163.

EXAMPLE 9 Selective and Nonselective Deprotection ofN-e-t-butoxycarbonyl-N-a-vinyloxycarbonyl-L-lysine The title compound isdissolved in methylene chloride and titrated with a dilute solution ofbromine in methylene chloride until the orange bromine color persists.Methanol is added and the solution allowed to stand at room temperaturefor an hour. All volatile material is stripped off at reduced pressure,leaving N- e-tBOC-L-lysine hydrobromide salt as a residue. An NMR ofthis residue in DMSO-d is identical with the NMR of authentic material.There are no vinyl protons present and no loss of the t-butoxycarbonylgroup. It is evident that by this procedure the VOC group is selectivelyremoved without touching the t-BOC group.

The title compound is treated with 1N HCl in dioxane for l hour and thevolatile substances then removed in vacuo. The product after standardworkup is identical with an authentic sample of L-lysinemonohydrochloride. By this procedure obviously both the t-BOC and VOCgroups have been split off.

EXAMPLE Use of Bromine to Remove VOC Residue in Production of HBr-HNGlyGlyOEt Vinyloxycarbonylglycylglycine ethyl ester is dissolved inmethylene chloride and then titrated with a dilute solution of brominein the same solvent until the orange bromine color persists. Thedibromide precipitates as the titration proceeds. A few ml methanol areadded to,the reaction mixture which is then left for an hour and finallyevaporated to dryness at reduced pressure. The crystalline residue isalready pure (it has the same M.P. 186-l87 as an authentic pure sampleof glycylglycine ethyl ester hydrobromide obtained by standardprocedures). The yield is quantitative and the m.p. does not change onrecrystallization.

EXAMPLE ll Use of Bromine to Remove VOC Residue in Production of HBI' 'HN-LPh8L-LeUOM Vinyloxycarbonyl-L-phenylalanyl-L-leucine methyl ester(2mM) is dissolved in 10 ml methylene chloride and then treated with 2mMof bromine. in the same solvent. Methanol (5 ml) is added to thereaction mixture which is then left at room temperature for two hoursand finally evaporated to dryness at high vacuum. The product isdissolved in acetone and the HBr salt precipitated by the slow additionof ether; 0.702g (94%). The dipeptide ester hydrobromide isrecrystallized from acetone-ether; M.P. l4ll42.5.

L-Alanylglycine ethyl ester hydrobromide (M.P. l44l46) andL-phenylalanylglycine ethyl ester hydrobromide (M.P. 136-137.5) aresimilarly prepared by deprotection of the vinyloxycarbonyl derivativeprecursors. Sometimes addition of a trace of HBr in ether aids in thecrystallization purification process.

EXAMPLE 12 Use of Mild Acid to Remove VOC Residue in Production of HCl-HN-L-Ala-GlyOEt Vinyloxycarbonyl-L-alanylglycine ethyl ester (1.0mM) isdissolved in 2 ml absolute ethanol and 2 ml ofa one molar solution ofhydrogen chloride in ethanol added. The reaction mixture is allowed tostand at room temperature for one hour and then the volatile substancesare stripped off in vacuo. The residue is triturated with ether yieldingessentially pure dipeptide ester hydrochloride (-99 percent) whichcrystallizes during the trituration process; M.P. l53l55.Recrystallization from ethanol/ether raises the melting point to l55l56.

The same product is obtained in high yield when the reaction is run indioxane. Glycylglycine ethyl ester hydrochloride (M.P. l85-l86) andL-asparaginylglycylglycine ethyl ester hydrochloride (hygroscopic solid)are obtained by closely related procedures from the respectivevinyloxycarbonyl derivatives.

EXAMPLE l3 Use of Mild Acid to Remove VOC Residue in Production of HBr'HN-L-Phe-L-LeuOMe Vinyloxycarbonyl-L-phenylalanyl-L-leucine methyl esterl .0 mM) is dissolved in 2 ml anhydrous methanol and 2 ml of a one molarsolution of HCl in anhydrous methanol is added. The reaction mixture isallowed to stand at room temperature for 2 hours and then is evaporatedat reduced pressure. The product HCl'l-l N L-Phe-L-LeuOMe is anamorphous solid. It is converted to the HBr salt by dissolution inwater, passing through an anion exchanger (Amberlite IRA-400) in thebromide form, and evaporation of the water. The residue isrecrystallized from acetone-ether; M.P. l4l-l42.

The same dipeptide ester hydrobromide can be prepared directly inessentially quantitative yield by treatment ofvinyloxycarbonyl-L-phenylalanyl-L-leucine methyl ester (1.0 mM) with 3ml of 1.0 molar HBr in acetic acid for ten minutes. The solventsstripped off and the product recrystallized as above; M.P. 141 .5l

EXAMPLE l4 Mercuric lon Induced Deprotection ofVinyloxycarbonyl-L-phenylalanine Vinyloxycarbonyl-L-phenylalanine (1.0g,4.25 mM) is dissolved in acetic acid and mercuric acetate (l.4g, 4.4 mM)added. The mixture is heated to 50 for 10 minutes and then most of theacetic acid is removed at reduced pressure. The residue is dissolved in50 ml water and to remove the mercuric ion, hydrogen sulfide is bubbledthrough the solution and the precipitated mercuric sulfide filtered off.The solution is taken to dryness and the residue recrystallized fromwater whose pH is manipulated to obtain maximum precipitation at theisoelectric point; yield 0.4g, 57%; M.P. 265 (dec). The filtrate afterisolation of this first crop contains by NMR analysis an additional 35%L- phenylalanine.

The same reaction occurs when the source of mercuric ion is mercuricchloride or mercuric nitrate.

The VOC group can be removed after an initial peptide condensation orthe protected peptide product can be used in further condensationprocedures prior to removal of the VOC group.

From the above it will be seen that the use of the VOC protecting groupprovides for the temporary blocking of a free amino group during peptidecondensation; with the blocking group being readily removed in highyields and under mild conditions and also under extremely selectiveconditions. ACCORDINGLY,

What is claimed is: 1. In a process for the synthesis of peptidescomprising reacting an a-amino acid, or a peptide built up from a-aminoacids, said a-amino acid or peptide built up from a-amino acids having aprotected N-terminal.

3. The improvement of claim 1, wherein said amino" group which isprotected with a vinyloxycarbonyl group is an a-amino group or ane-amino group.

4. In a process for the synthesis of peptides comprising reacting ana-amino acid, or a peptide built up from a-amino acids, said a-aminoacid or peptide built up from a-amino acids having a protectedN-terminal amino group, with an a-amino acid, or a peptide built up froma-amino acids, having a free amino group and a protected C-terminalcarboxyl group, wherein other functional groups in said a-amino acids orpeptides may be protected, the improvement comprising protecting anamino group with a vinyloxycarbonyl group and, after said reacting,removing said vinyloxycarbonyl group.

5. The improvement of claim 4 wherein said vinyloxycarbonyl group isremoved by reaction of the resulting vinyloxycarbonyl peptide with ahalogen or a pseudohalogen in a solvent, followed by addition of a loweralkanoland removal of the solvent and byproducts to produce theprotonated salt of the peptide.

6. The improvement of claim 5 wherein, after peptide condensation, thevinyloxycarbonyl group introduced during acylation is removed bytitration of the resulting vinyloxycarbonyl peptide with bromine in aninert solvent, followed by addition of methanol and removal of thesolvent and BrCH CH(OMe) at reduced pressure to produce the peptidehydrobromide.

2. The improvement of claim 1 wherein the vinyloxycarbonyl group isadded to said amino group by reacting with vinyl chloroformate.
 3. Theimprovement of claim 1, wherein said amino group which is protected witha vinyloxycarbonyl group is an Alpha -amino group or an epsilon -aminogroup.
 4. In a process for the synthesis of peptides comprising reactingan Alpha -amino acid, or a peptide built up from Alpha -amino acids,said Alpha -amino acid or peptide built up from Alpha -amino acidshaving a protected N-terminal amino group, with an Alpha -amino acid, ora peptide built up from Alpha -amino acids, having a free amino groupand a protected C-terminal carboxyl group, wherein other functionalgroups in said Alpha -amino acids or peptides may be protected, theimprovement comprising protecting an amino group with a vinyloxycarbonylgroup and, after said reacting, removing said vinyloxycarbonyl group. 5.The improvement of claim 4 wherein said vinyloxycarbonyl group isremoved by reaction of the resulting vinyloxycarbonyl peptide with ahalogen or a pseudohalogen in a solvent, followed by addition of a loweralkanol and removal of the solvent and byproducts to produce theprotonated salt of the peptide.
 6. The improvement of claim 5 wherein,after peptide condensation, the vinyloxycarbonyl group introduced duringacylation is removed by titration of the resulting vinyloxycarbonylpeptide with bromine in an inert solvent, followed by addition ofmethanol and removal of the solvent and BrCH2CH(OMe)2 at reducedpressure to produce the peptide hydrobromide.